Identification of a Cytoplasmic-Retention Sequence in ERK2

Abstract

A key step in the signaling mechanism of the mitogen-activated protein kinase/extracellular signal-responsive kinase (ERK) cascade is its translocation into the nucleus where it regulates transcription and other nuclear processes. In an attempt to characterize the subcellular localization of ERK2, we fused it to the 3′-end of the gene expressing green fluorescent protein (GFP), resulting in a GFP-ERK2 protein. The expression of this construct in CHO cells resulted in a nuclear localization of the GFP-ERK2 protein. However, coexpression of the GFP-ERK2 with its upstream activator, MEK1, resulted in a cytosolic retention of the GFP-ERK2, which was the result of its association with MEK1, and was reversed upon stimulation. We then examined the role of the C-terminal region of ERK2 in its subcellular localization. Substitution of residues 312–319 of GFP-ERK2 to alanine residues prevented the cytosolic retention of ERK2 as well as its association with MEK1, without affecting its activity. Most important for the cytosolic retention are three acidic amino acids at positions 316, 319, and 320 of ERK2. Substitution of residues 321–327 to alanines impaired the nuclear translocation of ERK2 upon mitogenic stimulation. Thus, we conclude that residues 312–320 of ERK2 are responsible for its cytosolic retention, and residues 321–327 play a role in the mechanism of ERK2 nuclear translocation. A key step in the signaling mechanism of the mitogen-activated protein kinase/extracellular signal-responsive kinase (ERK) cascade is its translocation into the nucleus where it regulates transcription and other nuclear processes. In an attempt to characterize the subcellular localization of ERK2, we fused it to the 3′-end of the gene expressing green fluorescent protein (GFP), resulting in a GFP-ERK2 protein. The expression of this construct in CHO cells resulted in a nuclear localization of the GFP-ERK2 protein. However, coexpression of the GFP-ERK2 with its upstream activator, MEK1, resulted in a cytosolic retention of the GFP-ERK2, which was the result of its association with MEK1, and was reversed upon stimulation. We then examined the role of the C-terminal region of ERK2 in its subcellular localization. Substitution of residues 312–319 of GFP-ERK2 to alanine residues prevented the cytosolic retention of ERK2 as well as its association with MEK1, without affecting its activity. Most important for the cytosolic retention are three acidic amino acids at positions 316, 319, and 320 of ERK2. Substitution of residues 321–327 to alanines impaired the nuclear translocation of ERK2 upon mitogenic stimulation. Thus, we conclude that residues 312–320 of ERK2 are responsible for its cytosolic retention, and residues 321–327 play a role in the mechanism of ERK2 nuclear translocation. mitogen-activated protein kinase dithiothreitol external signal-responsive kinase green fluorescent protein hemagglutinin myelin basic protein MAPK/ERK kinase peroxyvanadate nuclear export signal polymerase chain reaction Chinese hamster ovary polyacrylamide gel electrophoresis Mitogen-activated protein kinase (MAPK)1 signaling cascades are main routes of communication between the plasma membrane and regulatory intracellular targets and thus initiate a large array of cellular responses (1Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3218) Google Scholar, 2Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 3Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2292) Google Scholar, 4Cobb M. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (762) Google Scholar). The first MAPK cascade elucidated is the one that signals through the extracellular signal-responsive kinases 1 and 2 (ERK1/2), which are activated via a sequential phosphorylation and activation of the protein kinases Raf1 and MAPK/ERK kinase (MEK). Upon activation, ERK phosphorylates and activates several regulatory targets, which eventually culminate in regulation of proliferation, differentiation, and other cellular processes. Key steps in the signaling mechanism of the ERK cascade are the changes in localization of its components upon stimulation. In resting cells, all components of the cascade seem to be localized primarily in the cell cytosol. However, this localization is rapidly changed upon extracellular stimulation, which causes Raf1 recruitment to the plasma membrane (5Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (886) Google Scholar) and translocation of MEK (6Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar), ERK (7Chen R.H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar), and RSK (7Chen R.H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar) into the nucleus. After translocation, MEK seems to be rapidly exported from the nucleus by its nuclear export signal (NES; Ref. 8Fukuda M. Gotoh I. Gotoh Y. Nishida E. J. Biol. Chem. 1996; 271: 20024-20028Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), although the timing and role of its translocation are still controversial (9Fukuda M. Gotoh I. Adachi M. Gotoh Y. Nishida E. J. Biol. Chem. 1997; 272: 32642-32648Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (333) Google Scholar). ERK and RSK on the other hand are retained in the nucleus for longer times after stimulation, and this longer time is correlated with the effects of ERK on mitogenesis and neurite outgrowth in PC12 cells (11Traverse S. Gomez N. Paterson H. Marshall C. Cohen P. Biochem. J. 1992; 288: 351-355Crossref PubMed Scopus (807) Google Scholar,12Nguyen T.T. Scimeca J.C. Filloux C. Peraldi P. Carpentier J.L. Van-Obberghen E. J. Biol. Chem. 1993; 268: 9803-9810Abstract Full Text PDF PubMed Google Scholar). The mechanism of nuclear translocation of the different kinases is not fully understood. Recently, it was shown that in resting cells ERK is retained in the cytosol by its association with MEK (10Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (333) Google Scholar), and upon stimulation ERK is detached from this cytosolic anchor to rapidly translocate into the nucleus. This translocation requires dimerization and phosphate incorporation into the regulatory Thr and Tyr residues of at least one of the ERKs in the dimer (13Khokhlatchev A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). Here we further support the cytosolic retention of mammalian ERK2 by MEK1, which is reversed upon stimulation. We find that residues 312–320 of ERK2 play a crucial role in its MEK-induced cytosolic retention, and thus prevent nuclear localization of ERK in resting cells. We also provide evidence that residues 321–327 play a role in the nuclear translocation of ERK2. Thus, the 312–327 region of ERK2 plays an important role in securing the proper subcellular localization of ERK2 both before and after stimulation. Wild type ERK2 (bases 22-1096) was ligated into ApaI and XbaI sites downstream to green fluorescent protein (GFP) gene of pEGFP-C1 (CLONTECH). Mutations in ERK2 (GFP-312A, GFP-316A, GFP-321A, GFP-328A, GFP-ΔCOOH; see Fig. 1) were performed by PCR mutagenesis as described (14Silverman M.A. Benard O. Jaaro H. Rattner A. Citri Y. Seger R. J. Biol. Chem. 1999; 274: 2631-2636Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Wild type human MEK1 (MKK1) was ligated into BamHI and EcoRV sites of pCDNA1 (Invitrogen) as described (6Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar). The various plasmids were transfected into CHO cells using LipofectAMINE (Life Technologies, Inc.). Immunofluorescence studies were performed essentially as described (6Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar). Cells were stimulated with peroxyvanadate (VOOH; 100 μm Na3VO4/200 μmH2O2, 15 min), fixed (3% paraformaldehyde), and visualized by fluorescence microscopy (Zeiss Axioscop microscope, HBO 100 W/2; ×400 magnification). For HA-ERKs, cells were stained with anti-HA polyclonal antibody (1:100, Santa Cruz Biotechnology), and rhodamine-conjugated goat-anti-rabbit antibodies (1:100, Jackson ImmunoResearch). Transfected cells were serum-starved, stimulated, and lysed as described previously (6Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar). Activated ERK was detected by probing blots with anti-activated ERK monoclonal antibody (1:30,000, DP ERK, Sigma Israel). Total ERK protein was detected using either anti-ERK2 C-terminal antibodies (C-14, Santa Cruz Biotechnology) or anti-MAPK (Sigma, Israel). MEK1 was detected with anti-MEK1 monoclonal (Transduction Laboratories). Transfected CHO cells were serum-starved (24 h), stimulated (VOOH, 15 min), and harvested as described (6Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar). The cell extracts were then subjected to immunoprecipitation with anti-GFP monoclonal antibody (Roche Molecular Biochemicals). For MEK1 coimmunoprecipitation studies, the beads were washed as described (10Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (333) Google Scholar) with low stringency buffer (20 mmHEPES, pH 8.0, 2 mm MgCl2 2 mmEGTA) and then subjected to immunoblotting with monoclonal anti-MEK and anti-GFP antibodies. For the determination of ERK activity, the beads were washed once with 0.5 m LiCl, twice with radioimmune precipitation buffer, and with buffer A (50 mmβ-glycerophosphate, pH 7.3, 1.5 mm EGTA, 1 mmEDTA, 1 mm DTT, and 0.1 mm sodium vanadate) as described (14Silverman M.A. Benard O. Jaaro H. Rattner A. Citri Y. Seger R. J. Biol. Chem. 1999; 274: 2631-2636Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). GFP-ERK2 activity was measured as described previously (14Silverman M.A. Benard O. Jaaro H. Rattner A. Citri Y. Seger R. J. Biol. Chem. 1999; 274: 2631-2636Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In an attempt to characterize the subcellular localization of ERK2, we fused the cDNA of rat ERK2 to the 3′-end of the gene expressing GFP, resulting in a GFP-ERK2 protein. This fused protein was transfected into CHO cells and was examined for its subcellular localization using fluorescence microscopy. After serum deprivation that ensures the cytosolic distribution of endogenous ERK1 and ERK2 (data not shown), GFP-ERK2 was localized primarily in the nucleus (Fig. 2). As reported for Xenopus MAPK (10Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (333) Google Scholar), when MEK was coexpressed with GFP-ERK2, the subcellular distribution of GFP-ERK2 was different and appeared primarily in the cytosol. After stimulation with VOOH, a strong and non-selective activator of ERK, a large portion of GFP-ERK2 was translocated into the nucleus. Nuclear translocation was observed also with other stimuli (data not shown), although, a small portion (<15%) of GFP-ERK2 was observed in the cytosol under all conditions as described previously (15Reszka A.A. Seger R. Diltz C.D. Krebs E.G. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8881-8885Crossref PubMed Scopus (322) Google Scholar). Because the C-terminal region of ERK2 plays an important role in the localization and translocation of ERK2 (13Khokhlatchev A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 16Lenormand P. Sardet C. Pages G. L'Allemain G. Brunet A. Pouyssegur J. J. Cell Biol. 1993; 122: 1079-1088Crossref PubMed Scopus (587) Google Scholar), we modified several parts in this region (Fig. 1), which include (i) deletion of the C terminus of ERK2 (amino acids 338–358, ΔCOOH), (ii) substitution of amino acids 328–336 to alanines (328A), (iii) substitution of amino acids 321–327 to alanines (321A), (iv) substitution of amino acids 312–319 to alanines (312A), and (v) substitution of Asp-316, Asp-319, and Glu-320 to alanines (316A). As described for the GFP-ERK2, these constructs were fused to GFP and expressed in CHO cells, either with or without exogenous MEK1. The localization of ΔCOOH and GFP-328A appeared similar to that of GFP-ERK2 under all conditions and treatment used (data not shown). No changes as compared with GFP-ERK2 were detected also when the other three constructs 312A, 316A, and 321A, were expressed in CHO cells without additional MEK1 (Fig. 2). On the other hand, changes in localization were detected when these constructs were coexpressed with exogenous MEK1. Thus, unlike GFP-ERK2, GFP-312A was localized in the nucleus of resting cells, and this remained unchanged after VOOH stimulation. Nuclear localization was observed also with the GFP-316A protein, although the amount of this protein in the cytosol of resting cells was higher than that of the GFP-312A construct. GFP-321A had similar localization in resting cells as GFP-ERK2 when coexpressed with MEK1. However, stimulation with VOOH caused a uniform cellular distribution of this protein, indicating that this mutated protein might be compromised in its ability to translocate into the nucleus. To exclude the possibility that the altered localization is because of the GFP part of the constructs, we determined the localization of HA-ERK2 and HA-312A by staining with anti-HA antibody. Indeed, both constructs demonstrated similar distribution to that of their GFP counterparts (Fig. 2 B,left), indicating that the localization observed is indeed because of ERK2. Interestingly, when the amount of transfected GFP-ERK2 was significantly reduced, the weak staining observed was either cytosolic or spread all over the cell, even in the absence of exogenous MEK1 (Fig. 2 B, right). This cytosolic retention can be explained by an interaction of GFP-ERK2 with residual, unsaturated, cytosolic anchoring protein. Under these conditions, GFP-312A was still detected in the nucleus, indicating that the 312–319 region is responsible for this interaction. The subcellular localization of the various GFP-ERK mutants was further verified by isolating the nuclei of the GFP-ERK2 constructs-expressing CHO cells. As expected from the results in Fig. 2, Western blot analysis using anti-GFP antibodies revealed that the amount of GFP-ERK2 in the nucleus was reduced when coexpressed with MEK1 and elevated again upon stimulation (Fig. 3). The amount of GFP-312A and GFP-316A proteins in the nucleus was not significantly changed when MEK1 was coexpressed, whereas the amount of GFP-321A protein was reduced by MEK1 and only slightly enhanced after VOOH-stimulation. Together, these results show that GFP-312A, and to a lesser extent also GFP-316A, lost their MEK1-induced, cytosolic-retention in resting cells. On the other hand, GFP-321A protein exhibited a compromised VOOH-induced nuclear-translocation. Hence, residues 312–327 play an important role in the subcellular localization of ERK2 both before and after external stimulation. To clarify whether these differences in subcellular localization are because of altered ERK2 activation, we examined the phosphorylation state and catalytic activity of the various constructs. Thus, CHO cells were transfected with GFP-ERK2, GFP-312A, GFP-316A, and GFP-321A, either with or without MEK1. The cells were then lysed and immunoblotted with either anti-general ERK or anti-diphospho ERK antibodies. Beside the endogenous ERKs, an additional band of 70 kDa was detected by the antibodies, which represents the GFP-ERKs. Similar levels of expression of the various mutated proteins were detected by the anti-general ERK antibodies (Fig.4 A). Interestingly, no significant differences in the amount of phosphates incorporated into the regulatory Thr and Tyr residues were detected between the various mutants under any of the conditions used, indicating that the four GFP proteins undergo proper upstream activation. We then examined whether the various mutations affected the catalytic activity of ERK2. To do so, the various GFP-conjugated ERKs were immunoprecipitated with anti-GFP antibody and assayed for their ability to phosphorylate myelin basic protein (MBP). The activity of the mutants did not differ from that of GFP-ERK2, either with or without exogenous MEK1 (Fig.4 B). Therefore, these results indicate that residues 312–327 are not obligatory for either activation or catalytic activity of ERK2, and exclude the possibility that the altered localization of the modified proteins is because of compromised activation. Because the cytosolic localization of GFP-ERK2 seems to be MEK-dependent, we next asked whether this localization is because of an association between ERK and MEK and whether the mutations in the C terminus interfere with this ERK-MEK association. Lysates from CHO cells coexpressing MEK1 and the four GFP-ERK constructs were subjected to immunoprecipitation with anti-GFP antibodies. As expected (10Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (333) Google Scholar), immunoblotting with anti-MEK1 antibodies showed coprecipitation of MEK1 with GFP-ERK2, which was reversed upon VOOH stimulation (Fig.5). However, this coimmunoprecipitation was not stable and was easily disrupted, even under mild washing conditions (e.g. 0.15 m NaCl; data not shown). These results support the notion that, in resting cells, ERK and MEK are indirectly associated and dissociate upon stimulation to allow translocation of both MEK and ERK into the nucleus. On the other hand, no coprecipitation of MEK was detected in immunoprecipitates of GFP-312A either before or after stimulation. When GFP-316A was immunoprecipitated from resting cells, a small amount of MEK1 was coprecipitated, in agreement with the small amount of GFP-316A that remained in the cytosol in the fluorescence studies (Fig. 2). As with GFP-ERK2, VOOH reversed the coimmunoprecipitation of MEK by GFP-316A. GFP-321A pulled down MEK1 from resting cells and less from VOOH-stimulated cells, indicating that the association and dissociation of this mutant from MEK1 were not affected. As expected, both GFP-ERK2 and GFP-312A did not interact with a constitutively active MEK1 (Fig.5 B), indicating that the activation of these cells by active MEK was sufficient to prevent the ERK-MEK association. The mechanism by which ERKs are retained in the cytosol of resting cells and translocated into the nucleus is not fully understood. Based on our and others studies (10Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (333) Google Scholar, 16Lenormand P. Sardet C. Pages G. L'Allemain G. Brunet A. Pouyssegur J. J. Cell Biol. 1993; 122: 1079-1088Crossref PubMed Scopus (587) Google Scholar), it seems that ERK is kept out of the nucleus of resting cells by several anchoring proteins. One docking matrix is the microtubule cytoskeleton, which serves as a major docking matrix for up to 35% of the cellular ERK1 and ERK2 (15Reszka A.A. Seger R. Diltz C.D. Krebs E.G. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8881-8885Crossref PubMed Scopus (322) Google Scholar). Other putative anchoring proteins for ERK in the cytosol are MAP kinase phosphatases (MKPs) and in particular MKP3 (17Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssigur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (521) Google Scholar, 18Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. S. 1998; PubMed Scopus Google Scholar), these do not seem to be reversed by mitogenic the of ERK to and our results indicate that the cytosolic retention of ERK2 by MEK1 is reversed by mitogenic stimulation, thus the nuclear translocation of Similar results were previously ERK (10Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (333) Google Scholar), and we the also with ERK1 (data not shown). However, the amount of endogenous MEK1 seems to be significantly than that of ERKs in cells Biochem. Sci. 1996; Full Text PDF PubMed Scopus Google Scholar) and for the cytosolic retention of all ERK and MEK association is easily disrupted, and a small amount of ERK can be retained in the cytosol without exogenous is that additional proteins (e.g. the protein A. 1998; PubMed Scopus Google play a role in the ERK retention in the thus signaling which include than one ERK In this we examined the role of the C-terminal region of ERK in its subcellular localization. We that deletion of the C-terminal residues or substitution of residues 328–336 to alanines did not the subcellular localization of ERK2. However, it was reported (13Khokhlatchev A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar) that this C-terminal region a putative and A for the of of the mutations is that one of the mutations only part of the dimerization residues or the and the parts are still In to residues mutations in residues 321–327 affected nuclear translocation, and residues 312–320 affected cytosolic retention of The important residues for the cytosolic retention seem to be the three acidic residues Asp-316, Asp-319, and However, because the of GFP-316A was than that with GFP-312A, it is that additional residues between to the cytosolic Interestingly, Asp-319, which is to in was to be responsible also for the of ERK to M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. S. 1998; PubMed Scopus Google Scholar). However, because are proteins M. Chabert C. Muda M. Boschert U. Gillieron C. S. 1998; PubMed Scopus Google Scholar) and are not expressed in any significant amount in resting CHO cells (data not shown), this association is to for the cytosolic retention of ERK in resting cells. Upon mitogenic stimulation, ERK (7Chen R.H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar) as well as MEK (6Jaaro H. Rubinfeld H. Hanoch T. Seger R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3742-3747Crossref PubMed Scopus (167) Google Scholar, Shapiro P.S. S. Ahn N.G. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) are activated and translocate into the nucleus. to our this dissociation of these is that after dissociation from the anchoring protein, ERK to the nuclear membrane and then to the nucleus by an The of the endogenous ERK and MEK allow into the nucleus. However, it was that ERK a which its proper translocation (13Khokhlatchev A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar), and we show that the GFP-ERK2 can rapidly translocate into the nucleus. Thus, it is that the of these proteins and the of translocation of the endogenous ERK requires an active which might with or to the nuclear In we that substitution of residues 312–319 of ERK2 with alanine residues not its activity, the cytosolic retention of ERK, as well as its association with MEK1. Substitution of residues 321–327 to alanines the nuclear translocation of ERK2 upon mitogenic stimulation. We conclude that residues 312–327 of ERK2 play a role in its subcellular localization both before and after stimulation.